The invention relates to methods and devices used for digesting small amounts of protein in tiny cut gel pieces and for extracting the peptides resulting from the digestion in preparation for analysis by mass spectrometry.
The invention involves digesting proteins using enzymes within the gel pieces in vessels which have permeable but lyophobic bases in such a manner that they scarcely touch the walls of the vessel, and then rapidly removing the digested proteins from the gel pieces almost completely by gentle centrifuging. It is then advantageous to bond the peptides reversibly to suitable surfaces as quickly as possible. For this purpose, the bases of the vessels may contain structures for bonding the peptides which are suitable for washing and subsequently eluting the peptides. A number of vessels can be combined together to form plates which, for example, can have the size of microtiter plates.
Two-dimensional gel electrophoresis is still one of the best and most widely used methods for separating the proteins of a cell aggregatexe2x80x94the so-called xe2x80x9cproteomexe2x80x9d. When used after enzymatic digestion of proteins to peptides, mass spectrometry is the most sensitive methods for identifying individual proteins and determining their structure. The method itself and the difficulties encountered in using it will be briefly described in the following.
After separation in the gel, the proteins are stained and small samples of the gel containing the protein of interest are cut or punched out around the stain site. The gel samples are placed in a vessel and the stain is removed. Topping up with an enzyme solution (such as trypsin) leads to selective digestion at the cleavage sites determined by the enzyme. When using trypsin, which cleaves the molecule at two specific amino acids, the digestion produces peptides with a broad molar mass distribution around an average of approx. 1000 atomic mass units. The protein is usually clearly characterized by the exact masses of the peptides produced by the digestion. The peptides are able to diffuse inside the gel and slowly migrate out of the gel into the surrounding liquid within a few hours.
The peptides in the fluid are purified and analyzed by a suitable method of analysis using mass spectroscopy. In this method, the molecules are usually ionized by so-called Matrix Assisted Laser Desorption Ionization (MALDI) and the precise masses of the peptides resulting from digestion are measured in a time-of-flight (TOF) mass spectrometer. Other methods of ionization are known and are used, usually with confirmation from other types of mass spectrometer. For the MALDI-TOF analysis, the peptides produced by digestion are introduced on suitable sample carriers into small crystals of matrix substances and bombarded with laser pulses in a mass spectrometer. Their precise masses are determined by the flight time of the ions in the TOF mass spectrometer.
Even if some of the peptides are lost during the processing stage, precisely measured values of the peptides which are still available usually result in clear identification of the protein when a suitable program is used to search the protein sequence databases. It is possible to clarify ambiguous identifications by extensive measurement of the fragments of individual peptides produced by using special methods and elucidating them via their internal structure. This form of identification means that the protein, if known, can be characterized by its name, code, origin and molecular structure.
Details on the measurement of fragmentation and other methods for de-novo sequencing of proteins will not be given here.
Although such a straightforward method for the identification of proteins has been the subject of interest for the past few years, interest is now increasingly being directed toward the differences found between the proteins examined and those in the database. These differences, which relate to the mutative or post-translational changes in the proteins, are and will be the focus of interest in the future. For this, it is not only necessary to be able to measure only a few peptides, which in most cases is sufficient for identification purposes alone, but all possible peptides produced by the digestion. In the methods briefly described above however, many peptides are lost due to their being adsorbed on the walls of the vessel which contains them.
At the same time, it is not only the very concentrated proteins which are present in the gel at concentrations ranging from 10 to 100 picomol which are of interest, but also those at lower concentrations ranging from 10 to 100 femtomol. Now if, for example, 20 femtomol of a peptide is present in 20 microliters of liquid, the individual molecules produced by the digestion process will diffuse freely throughout the liquid and will come into contact with the walls of the vessel many times over within a matter of hours. In a small vessel of around three millmeters diameter, they will come into contact with a wall surface area of around 40 mm2. If the wall selectively adsorbs one of the peptides, then that peptide will cover the whole surface with a layer which is at least molecule deep. This monomolecular layer could adsorb approx. 40 picomol of a peptide of 1,000 atomic mass units, i.e. 2,000 times the amount which is available in the solution in our example. Even if the adsorptivity of the vessel wall could be reduced to one thousandth by taking the appropriate measures, the peptide of interest could still be fully adsorbed onto the surface and, if the adsorption bond is irreversible as is frequently the case, there is no way that the peptide could be brought into solution again.
The 20 microliter solution sample used in the example calculated here is currently regarded as large. Converting this sample size to a 1 microliter or 100 nanoliter sample (for the same concentration of test molecule) would increase the effect of coming into contact with the wall dramatically.
In modern biochemistry and molecular biology, samples are typically processed in large numbers simultaneously. A visible exponent of this development is the so-called microtiter plate consisting initially of 96, then 384 and now 1,536 reaction vessels. Recently, a NanoWell(trademark) plate was introduced with 3,456 reaction vessels. Increasing the number still further and providing the tools for processing is only a question of time. Appropriate pipetting and processing robots with storage positions for many microtiter plates, barcode labeling, multi-pipette heads and multi-dispensing systems have been developed for microtiter plates which have been in common use until now.
At the same time, the quantities of sample molecules required for the chemical, enzymatic and analytical processing stage are getting increasingly smaller so that proteins at very low concentration can also be measured. Processing has long since moved from the nanomol range to the picomol, femtomol and even attomol range. The disadvantage of this is that, as the amounts of test solution are progressively reduced, the wall area of the cavities enclosing the liquid progressively increases in relation to their volume and the chemical and physical effects of the cavity walls on the reactions during processing become more critical.
The microtiter plate also forms the ideal basis for processing the proteins of a proteome, for example, in association with an automated gel sampler. Until now, however, microtiter plates have whisked away a large proportion of the peptides produced by the digestion.
The objective of the invention is to find methods and devices to be used for preparing small samples of protein from pieces of gel for analysis using mass spectrometry but which are characterized by high yields for all peptides and particularly low levels of peptide loss for those peptides which are at risk through wall adsorption.
In detail, the method of the invention uses tiny gel pieces containing protein and relates to the enzymatic digestion of proteins within the gel which involves the following steps:
1) The gel pieces containing the proteins are placed in vessels with lyophobic porous bases.
2) Enzyme solution is added to the gel pieces but only as much as can be absorbed by them.
3) The proteins in the gel pieces are digested.
4) The vessels with the gel pieces are centrifuged so that the enzyme solution containing the peptides produced by the digestion are forced out of the gel pieces and centrifuged out of the vessels.
It is particularly convenient if the peptides in the enzyme solution which has been forced out of the gel pieces are immediately and reversibly adsorbed onto peptide-adsorptive surfaces so that they are no longer available for uncontrolled wall adsorption.
In this case, a lyophobic surface is defined as a surface which is not only water repellent (i.e. not only hydrophobic) but is also repellent to the organic solvents used in peptide chemistry, such as methyl alcohol or acetonitrile at least in aqueous solutions over a wide concentration range.
The device according to the invention consisted of providing the vessel with a lyophobic porous base where the porosity of the base is achieved by using one or more lyophobic capillary channels. These can be closed off with fibrous membranes, close-packed particle structures, fritt-type structures or open-pore solid foams. A number of vessels, each with porous bases, can be combined together in a single plate the size, for example, of a microtiter plate. By specially preparing their surfaces to give them the necessary adsorption properties, these membranes, close-packed particle structures or solid foams can be used to bond to and immobilize the peptides. The capillary channels in the bases of the vessels are made lyophobic so that the repulsive effect of capillary action prevents the liquids which have been freshly placed in the vessels from flowing out of the vessels under normal gravitational force but allows them to escape under centrifugal force. The internal surfaces can also be made lyophobic so that liquids can rapidly coalesce on the surfaces and rapidly escape under the effect of centrifugal force. Apart from this, surfaces which have been made lyophobic cause the level of contact with the wall to diminish since liquids touching the wall will not spread and wet the surface.
Basically, according to the invention, the purpose of the method is to keep the molecular contact between the dissolved peptide and the wall, which is proportional to the product of the area of the wall in contact with the solution and the duration of contact, as low as possible. After the proteins have been digested, the peptides bond to suitable surfaces as rapidly and reversibly as possible so that they have to be kept away from any further, uncontrolled contact with the wall; this process is promoted by the device according to the invention. The required reversible bonding at suitable surfaces allows the peptides to be subjected to further processes, such as washing, and the reversibility of the bond allows the peptides to be measured using mass spectrometry, for example, by transferring them to suitable mass-spectrometric MALDI sample carriers.
It is particularly beneficial to free the gel pieces of a part of their internal liquid before or after filling the vessels in Step 1) of the method. Some of the internal liquid can be removed from the gels, for example, after depostion in the vessels by centrifuging, or better still, by partial vacuum drying. Vacuum drying gives the gel an open-pore structure, which is good for the absorption of the enzyme solution in Step 2), but the gels must not be allowed to dry completely as this will impair their ability to re-absorb the liquid.
The enzyme solution can be added in Step 2) by the method of pipetting, using multiheaded pipettes if necessary, or by contactless dispensing using Piezo or solenoid dispensers. The absorption of enzyme solution due to the swelling of the partially dried, porous gel pieces carries the enzymes rapidly and uniformly into the gel pieces which, because the enzymes must be able to migrate by diffusion, is not the case with gels which have not been partially dried. After the enzyme solution has been absorbed by the gel pieces, surface contact of the solution with the wall of the vessel is minimal. Since the diffusion of the peptides produced in Step 3) is widely limited within the gel pieces, losses due to wall adsorption are negligibly small.
The digestion in Step 3) of the method is accelerated by warming the vessel (incubation) to the digestion temperature and, at the optimum temperature, the digestion process is complete within two to four hours. Digestion is therefore the rate-determining step. During the incubation period, the vessels must be well sealed to prevent the gel pieces from drying out.
In Step 4), the centrifugal force drives the enzyme solution with the peptides produced by the digestion out of the gel pieces within a few seconds and they immediately may drop through the porous bases of the vessels into a set of collector vessels or onto mass-spectrometric sample carriers underneath. Very moderate centrifuging conditions at approx. 2,000 rpm are sufficient.
After or during this process, the peptides may bond to the surfaces either on the porous structures of the base of the vessel or on the particles which are in the collector vessel or on the sample carrier plates. The particles in the collector vessels may, for example, be tiny magnetic beads with suitably prepared surfaces; these can be held or moved back and forth in a predetermined manner through the washing liquids by magnetic forces. Areas on the surface of sample carriers can be coated with adsorptive layers such as C18 alkyl chains to facilitate subsequent washing.
After the process has been completed in accordance with the invention, the peptides bonded to the surface can be washed in the usual manner and freed from enzymes, buffers, salts, and all remaining impurities. It is possible to release the peptides from the surface using an eluant solution such as 30% acetonitrile in de-ionized water and analyze the solution by mass spetrometrometry using, for example, sample carriers used for mass spectrometry.
It is also possible to place the peptides directly on MALDI sample-carrier plates during centrifuging without adsorptive bonding, for example, on plates prepared beforehand with MALDI matrix substances. In this method, it is better if alkaline metal salts are not used for the buffer solution. These can be substituted by ammonium carbonate.